In Vitro RNA Splicing in Mammalian Cell Extracts

Nanette Rooke1, Jason Underwood1

1 University of California, Los Angeles, Los Angeles, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.17
DOI:  10.1002/0471143030.cb1117s18
Online Posting Date:  May, 2001
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Abstract

Almost every eukaryotic pre‐mRNA generated by RNA polymerase II transcription requires the removal of introns to create mRNA. The correct splicing of constitutive exons is thus critical for normal protein expression and function. Moreover, the removal of many introns by the spliceosome is controlled in a tissue‐specific or developmentally specific manner. In order to study RNA splicing at a biochemical level, it is necessary to employ an in vitro, or cell‐free, system. Cell‐free splicing systems require two main components: (1) an extract made from mammalian cell nuclei and (2) the introns and exons of the eukaryotic gene of interest. This minigene construct allows the synthesis of sufficient quantities of pre‐mRNA substrates in vitro, which are then incubated in the nuclear extract and analyzed for splicing. Nuclear extracts, first developed for studying transcription in vitro, are modified for splicing. This unit describes how to set up an in vitro splicing reaction using a mammalian nuclear extract derived from either cell line or tissue, and how to analyze the splicing reaction products.

     
 
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Table of Contents

  • Creating a Minigene Construct for Testing a Novel Substrate
  • Basic Protocol 1: In Vitro Splicing of Pre‐Messenger RNA
  • Basic Protocol 2: Analysis of Splicing Reaction RNA Products
  • Support Protocol 1: Preparation of Nuclear Extract from Tissue Culture Cells
  • Support Protocol 2: Preparation of Nuclear Extract from Primary Tissue
  • Support Protocol 3: In Vitro Transcription of Capped, Radiolabeled RNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Vitro Splicing of Pre‐Messenger RNA

  Materials
  • Buffer DG (see recipe)
  • 10 mM ATP
  • 0.5 M creatine phosphate
  • 55 mM MgCl 2
  • 20% (w/v) polyethylene glycol 3350 (PEG; mol.wt. 3350; Sigma)
  • Nuclear extract (Support Protocols protocol 31 and protocol 42)
  • 20 U/µl RNase inhibitor (RNA guard; Amersham Pharmacia Biotech; Ribonuclease Inhibitor, porcine)
  • 5 to 20 fmol/µl uniformly radiolabeled pre‐mRNA splicing substrate ( protocol 5)
  • 20 mg/ml proteinase K (Amersham Pharmacia Biotech)
  • 10% (w/v) sodium dodecyl sulfate (SDS; appendix 2A)
  • 1.1× PCA buffer (see recipe)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (PCA)
  • 20 mg/ml glycogen (Roche)
  • 100% ethanol, ice‐cold
  • RNA loading buffer (see recipe)
  • 30°C and 80°C water baths or heating blocks
  • Whatman 3MM filter paper
  • Kodak XAR X‐ray film (standard 14 × 17–in.) with intensifying screen, or phosphor imager screen and imager (e.g., PhosphorImager from Molecular Dynamics or equivalent; see unit 6.3)
  • Additional reagents and equipment for denaturing PAGE of nucleic acids ( appendix 3A) and autoradiography (unit 6.3)

Basic Protocol 2: Analysis of Splicing Reaction RNA Products

  Materials
  • ∼5–8 × 109 HeLa cells (∼12 liters of suspension culture grown in spinner flasks; roller bottles are utilized for some cell types) or other mammalian cell of interest, in suspension or monolayer culture
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Phosphate‐buffered saline (PBS; appendix 2A) with 1 mM EDTA
  • Buffer A (see recipe)
  • Buffer C420 (see recipe)
  • Buffer DG (see recipe)
  • Beckman centrifuge with JS‐4.2 and JA‐20 rotors (or equivalents) and appropriate centrifuge tubes/bottles
  • 50‐ml (or other appropriate size) conical tubes, graduated
  • Rubber policeman or cell scraper
  • 40‐ml Dounce homogenizer with tight (type A) and loose (optional) pestle (Wheaton)
  • Dialysis tubing (10,000 to 14,000 MWCO), prepared as described in appendix 3C
  • Conductivity meter (optional)
  • Liquid nitrogen
  • Additional reagents and equipment for trypan blue staining (unit 1.1), dialysis ( appendix 3C), and Bradford protein assays ( appendix 3H)
NOTE: Perform all steps of this procedure at 0° to 4°C. All buffers, vessels, and centrifuge rotors should be precooled to this temperature.

Support Protocol 1: Preparation of Nuclear Extract from Tissue Culture Cells

  Materials
  • Sucrose cushion buffer (see recipe)
  • Dissected whole brains from six 28‐day Sprague‐Dawley rats
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Homogenization buffer (see recipe)
  • Buffer C230 (see recipe)
  • Buffer DG (see recipe)
  • Dissection tools
  • Small Petri dish or tissue‐culture plate
  • Beckman SW 28 rotor (or equivalent) and polyallomer ultracentrifuge tubes
  • Motor‐driven Teflon‐glass homogenizer (e.g., Wheaton Overhead Stirrer; Wheaton Science Products)
  • Small dialysis cassette (0.1 to 0.5 ml Pierce “Slide‐a‐lyzer” or equivalent), 10,000 MWCO (also see appendix 3C)
  • Additional reagents and equipment for dialysis ( appendix 3C), trypan blue staining (unit 1.1), and Bradford protein assays ( appendix 3H)
NOTE: Chill homogenization buffer and glass‐Teflon homogenization apparatus overnight at −20°C before the procedure and perform all steps at 0° to 4°C. All other buffers, vessels and centrifuge rotors should be precooled. The volumes in this procedure assume a starting material of six whole brains from 28‐day rats, ∼15 grams of tissue. For other tissues, this protocol should be scaled according to the mass of starting material.

Support Protocol 2: Preparation of Nuclear Extract from Primary Tissue

  Materials
  • RNase‐free water ( appendix 2A)
  • 10× transcription buffer (provided with polymerase, or see recipe)
  • 0.1 M DTT
  • NTP mix (see recipe)
  • Cap analog solution (see recipe)
  • 12.5 µM [α‐32P]UTP (800 Ci/mmol)
  • 0.1 µg/µl DNA template (linearized plasmid or PCR product) in 10 mM Tris⋅Cl, pH 8.1 (see appendix 2A for Tris buffer)
  • 10 U/µl bacteriophage RNA polymerase (T3, T7, or SP6)
  • RNase‐free DNase I (if PCR product is used as template)
  • Formamide loading buffer (see recipe)
  • RNA elution buffer (see recipe)
  • 70% and 100% ethanol, –20°C
  • TE‐acetate buffer (see recipe)
  • 85°C heating block
  • Scintillation counter and vials appropriate for Cerenkov counting
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis ( appendix 3A) and autoradiography (unit 6.3)
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Figures

Videos

Literature Cited

Literature Cited
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   Bewsey, K.E., Huff, J.P., and Johnson, M.E. 1991. Rapid isolation and purification from agarose gels: The phenol freeze‐fracture method. Biotechniques. 10:724‐725.
   Black, D.L. 1992. Activation of c‐src neuron‐specific splicing by an unusual RNA element in vivo and in vitro. Cell 69:795‐807.
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   Sierra, F., Tian, J.M., and Schibler, U. 1993. In vitro transcription with nuclear extracts from differentiated tissues. In Gene transcription: A practical approach (B.D. Hames and S.J. Higgins, eds.), pp. 125‐152. Oxford University Press, New York.
   Takagaki, Y., Ryner, L.C., and Manley, J.L. 1988. Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre‐mRNA polyadenylation. Cell 52:731‐742.
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   Zhou, Z., Luo, M.J., Straesser, K., Katahira, J., Hurt, E., and Reed, R. 2001. The protein Aly links pre‐messenger RNA splicing to nuclear export in metazoans. Nature 407:401‐405.
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